WO1999003823A2 - Nouveaux analogues de polyamine utilises comme agents therapeutiques et diagnostiques - Google Patents

Nouveaux analogues de polyamine utilises comme agents therapeutiques et diagnostiques Download PDF

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WO1999003823A2
WO1999003823A2 PCT/US1998/014896 US9814896W WO9903823A2 WO 1999003823 A2 WO1999003823 A2 WO 1999003823A2 US 9814896 W US9814896 W US 9814896W WO 9903823 A2 WO9903823 A2 WO 9903823A2
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polyamine
composition according
group
alkyl
cells
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PCT/US1998/014896
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English (en)
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WO1999003823A3 (fr
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Nicolaas M. J. Vermeulin
Christine L. O'day
Heather K. Webb
Mark R. Burns
Donald E. Bergstrom
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Oridigm Corporation
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Priority to JP2000503054A priority Critical patent/JP4044728B2/ja
Priority to CA002297413A priority patent/CA2297413A1/fr
Priority to AU84968/98A priority patent/AU758570B2/en
Priority to US09/341,400 priority patent/US6172261B1/en
Priority to EP98935790A priority patent/EP1001927A2/fr
Publication of WO1999003823A2 publication Critical patent/WO1999003823A2/fr
Publication of WO1999003823A3 publication Critical patent/WO1999003823A3/fr
Priority to US09/396,523 priority patent/US7208528B1/en
Priority to US09/713,512 priority patent/US7160923B1/en

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    • C07C233/36—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
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    • C07C233/78—Carboxylic acid amides having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by amino groups with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by an acyclic carbon atom
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    • C07C235/20—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton being acyclic and saturated having at least one of the singly-bound oxygen atoms further bound to a carbon atom of a six-membered aromatic ring, e.g. phenoxyacetamides having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C235/32—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings
    • C07C235/34—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to acyclic carbon atoms and singly-bound oxygen atoms bound to the same carbon skeleton the carbon skeleton containing six-membered aromatic rings having the nitrogen atoms of the carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
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    • C07C235/44—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring
    • C07C235/50—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by oxygen atoms having carbon atoms of carboxamide groups bound to carbon atoms of six-membered aromatic rings and singly-bound oxygen atoms bound to the same carbon skeleton with carbon atoms of carboxamide groups and singly-bound oxygen atoms bound to carbon atoms of the same non-condensed six-membered aromatic ring having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C237/04—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
    • C07C237/10—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated having the nitrogen atom of at least one of the carboxamide groups bound to an acyclic carbon atom of a hydrocarbon radical substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C237/20—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
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    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
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    • C07C237/22—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton having nitrogen atoms of amino groups bound to the carbon skeleton of the acid part, further acylated
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    • C07C271/00—Derivatives of carbamic acids, i.e. compounds containing any of the groups, the nitrogen atom not being part of nitro or nitroso groups
    • C07C271/06—Esters of carbamic acids
    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/20—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by nitrogen atoms not being part of nitro or nitroso groups
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    • C07C271/08—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms
    • C07C271/10—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C271/22—Esters of carbamic acids having oxygen atoms of carbamate groups bound to acyclic carbon atoms with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5308—Immunoassay; Biospecific binding assay; Materials therefor for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites

Definitions

  • the invention in the field of chemistry and biochemistry relates to the synthesis and use of novel polyamine transport (PAT) inhibitor compounds with pharmacological or agricultural uses and as probes for biochemical assays or for purification of selected polyamine binding targets.
  • PAT polyamine transport
  • these compounds are used to treat disorders of undesired cell proliferation, primarily cancer, alone or combined with other agents such as polyamine synthesis inhibitors.
  • An assay employing some of these compounds is useful for monitoring polyamine uptake or transport (PAT) and allows analysis of binding sites for polyamines or for other basic ligands on a variety of molecules.
  • This invention also relates to the synthesis and use of novel polyamine combinatorial libraries.
  • These libraries are used to discover compositions that inhibit PAT and/or that bind to a cellular polyamine transporter (PATr).
  • PATr cellular polyamine transporter
  • Various members of these libraries or compounds discovered through use of the libraries have utility as drugs, agricultural chemicals, and as probes.
  • This invention also identifies key elements that comprise the polyamine binding sites of membrane as well as soluble proteins.
  • Polyamines are ubiquitous molecules that provide a "buffer" system for the cell by modulating the activities of proteins, RNA, DNA, and lipids. Polyamines may play a direct role in apoptosis. Mammals and other organisms have an active polyamine uptake and recycling system that complements their polyamine synthetic capabilities. Because polyamines modulate such a large range of molecules and cellular activities, polyamine analogues, as disclosed herein, offer novel approaches for targeting a variety of disease states, particularly cancer, and also provide unique tools to monitor cellular activities. Polyamines and Cancer
  • polyamines as anticancer agents.
  • Polyamines affect chromatin structure in eukaryotes and prokaryotes by binding specifically to DNA (Balasundaram, D. et al, Mol. Cell. Biol. 100:129-140, 1991) so that condensation occurs when the binding sites on DNA are saturated.
  • Acetylation of polyamines and histones lowers their affinity for DNA and is believed to occur in tandem to alter the structure and function of the nucleosome, thus regulating DNA replication and transcription by loosening DNA at the ends of the core particle.
  • polyamines are absolutely essential for DNA replication, they are of interest in the treatment of cancer. Particular interest has been focused on preventing cell proliferation by lowering intracellular polyamine levels.
  • Polyamine analogues as described herein are useful for preventing or treating cancer and other proliferative diseases by acting at a number of different levels.
  • the present invention focuses on the inhibition of PAT.
  • Other targets include induction of spermine/spermidine acetyltransferase
  • SSAT hypusine modification
  • other proteins that inhibit the cell cycle or induce apoptosis.
  • mice harboring wild-type L1210 cells with a combination of (1) DFMO (2) a low polyamine diet and (3) antibiotics (which decrease polyamine production by gut flora) resulted in prolonged survival compared to treatment with DFMO alone.
  • mice infected with a multi-drug resistant strain of malaria were cured by treatment with a chloroquinoline-putrescine conjugate (Singh, S. et al, J. Biol. Chem. 272:13506-13511, 1997).
  • cytotoxic compounds could be enhanced by their conjugation with polyamines.
  • These effects may have been due to the exploitation of the PAT system to deliver these compounds into cancer cells.
  • the present invention is therefore directed in part to rapid and efficient testing of many different conjugates between polyamines and known drugs for their transport into cells.
  • this invention combines the cytotoxic properties of known drugs with the facilitated transport of polyamines, which relies on the present inventors' discoveries surrounding the PATr described herein.
  • S ARs structure-activity- relationships
  • PAT Polyamine Transport Assays
  • a radiochemical assay is used for biochemical analysis of transport and has been used to study PAT in yeast and a variety of mammalian cells (Kakinuma, Y. et al, Biochem. Biophys. Res. Comm. 216:985-992, 1995; Seiler, N. et al, Int. J Biochem. Cell Biol. 28:843-861, 1996). See, for example Huber, M. et al. Cancer Res. 55:934-943, 1995.
  • the radiometric assay uses radiolabeled polyamines such as putrescine, spermidine or spermine, but, due to the low signal, large numbers of adherent or non-adherent cells are required. Additional care is required with spermine due to its non-specific adsorption to cells and plastics.
  • Cells are mixed with the test compounds and the radiolabeled polyamine to initiate the assay. The cells are incubated for 1 -60 minutes, depending on cell type. The assay is terminated by removal of the medium and cooling the plates to 4°C. The cells are then washed with cold medium three times, dissolved in 0.1% sodium dodecyl suifate and the radioactivity in solution is then determined by scintillation counting. This assay is difficult to scale up to a high throughput procedure due to the low signal from the radiolabel and the handling requirements inherent in procedures with radioactivity.
  • SSAT Spermine/Spermidine Acetyltransferase
  • the protein eIF-5A appears to play a role in protein synthesis, although its exact function remains obscure (Hanauske-Abelm, H. M. et al, FEBS Lett.
  • EIF-5A is unique in that it is modified by the unusual amino acid hypusine. Hypusine is generated post-translationally by the sequential action of deoxyhypusyl synthase (using spermidine as a substrate) and deoxyhypusyl hydroxylase. Inhibition of this modification of eIF-5A coincides with proliferative arrest late in the Gl phase of the cell cycle. This modification occurs in most, if not all, eukaryotes. The present inventors have noted that inhibitors of deoxyhypusyl synthase would be useful in treating diseases associated with unwanted cell proliferation, such as cancer, by blocking the cell cycle.
  • Inhibition of Angiogenesis decreases the vascularization of solid tumors.
  • One month of treatment with DFMO resulted in a 50% reduction in neoplastic vessel count in humans with cervical interepithelial neoplasia (Mitchell, M.F. et al., Proceedings AACR 39:Ab. 600, 1998).
  • DFMO inhibited the neovascularization induced by tumor cells in vivo (Jasnis, M.A. et al, Cancer Lett 79:39-43, 1994).
  • Squalene a polyamine analogue, also inhibits angiogenesis in the rabbit cornea assay.
  • CHENSpm is polyamine analogue that induces apoptosis but does not function through any of the mechanisms described above (Ha, H.C. Proc Nat.Acad.Sci USA 94:11557- 11562(1997) CHENSpm does not induce SSAT, but does reduce spermidine and spermine levels and produces a G 2 cell cycle arrest at subtoxic concentrations, suggesting an unusual mode of action.
  • Polyamines are known to bind to tubulin and promote its bundling, though this is just one of several possible mechanisms by which polyamines can induce apoptosis or inhibit cell growth.
  • MAPs microtubule associated proteins
  • Acetylated polyamines the products of the spermine/spermidine acetyltransferase (SSAT) enzymatic reaction, are substrates for the enzyme polyamine oxidase. Oxidation of acetylated polyamines produces a stoichiometric release of H 2 O 2 which is believed to be responsible for the apoptotic response. This induction is cell type-specific and is believed to be due to the accumulation of the polyamine analogue in the cell and its possible binding to a polyamine-sensitive repressor or activator of transcription of SSAT. This repressor has not been identified, but a probe/assay for its detection would enable the synthesis of better drugs.
  • Membrane-bound Proteins the products of the spermine/spermidine acetyltransferase (SSAT) enzymatic reaction. Oxidation of acetylated polyamines produces a stoichiometric release of H 2 O 2 which is believed to be
  • polyamine binding receptors such as calcium receptor, N-methyl-D- aspartate (NMD A) receptors, glutamate receptors, Ca 2+ channels and several of the inwardly rectifying K + channels (Ventura, C. et al, Am. J. Physiol
  • PAT inhibitors can contribute to inhibition of cell growth, they are viewed by the present inventors as being useful in the treatment of post- angioplasty injury. Endothelial denudation and vessel wall injury lead to neointimal hyperplasia and luminal stenosis. Inhibition of smooth muscle cell proliferation, for example, could inhibit neointimal formation. According to this invention, this initiation of cell proliferation after injury is amenable to treatment with PAT inhibitors preferably in combination with polyamine synthesis inhibitors (Takagi, M.M. et al, Arlerioscler. Thromb. Vase. Biol. 17:3611-3619, 1997; Nakaoka, T. et al, J. Clin. Invest. 100:2824-2832, 1997; Maillard, L. et al, Cardiovasc. Res. 35:536-546, 1997).
  • Ca 2+ channels which have high affinity binding sites for polyamines, are modulated by polyamine levels.
  • Polyamines modulate the ⁇ -adrenergic-mediated changes in Ca 2+ levels and contractility (Ventura, C. et al., Am. J. Physiol 267: H587-H592, 1994).
  • Ca 2+ channels and binding can be measured as described for the NMDA receptor and the K + -inward rectifying channels in Ventura, supra.
  • an appropriate polyamine or analogue can be harnessed to modulate Ca 2+ in place of the channel blockers currently in use.
  • Ca 2+ sensing receptor located in the parathyroid and kidney.
  • the CaR has a specific polyamine binding site. Modulation of this receptor is believed to be a promising approach to the treatment of osteoporosis.
  • the CaR plays a different role in the brain from that in the parathyroid. Aggregated ⁇ -amyloid protein in this disease can stimulate the CaR and eventually lead to its down-regulation. Polyamines, likewise, can bind to the CaR and inhibit CaR down-regulation stimulated by ⁇ -amyloid. Polyamines or polyamine analogues can therefore serve as protective molecules.
  • RA rheumatoid arthritis
  • AdoMet S-adenosylmethionine
  • RA, and IL-2 is low in patients' synovial fluid, a condition which was reversed by inhibitors of polyamines (Flesher, E. et al, J. Clin. Invest. 83:1356-1362, 1987).
  • the polyamine synthesis inhibitor DFMO prolongs the life of MRLApr/lpr mice, a model of systemic lupus erythematosus.
  • Polyamine levels are elevated in the urine, synovial fluid, synovial tissue, and peripheral blood mononuclear cells of RA patients. Culturing these cells in the presence of methotrexate inhibited the production of IgM-rheumatoid factor. Spermidine reversed this effect, indicating to the present inventors that a combination of polyamine synthesis inhibitors and PAT inhibitors are useful can treat autoimmune diseases.
  • Other polyamine analogues, spergualin and deoxyspergualin are immunosuppressive and may be beneficial for treating multiple sclerosis (Bergeron et al., J Org. Chem. 52:1700, 1987; Drug Fut. 76:1165, 1991).
  • DNA/RNA-Polyamine Hybrids for Stable Binding to Nucleic Acids The spacing between ammonium polycations in naturally occurring polyamines (spermidine and spermine) is 3-4 carbons. This is the exact spatial separation for optimal binding to a DNA or RNA polyanionic phosphate backbone. It has been suggested that this interaction, together with the interaction with chromatin proteins, modulates gene transcription and expression. Recently, such ionic interactions have been exploited by combining polycationic 3', 5'- polyguanidine linkers bound with the base portion of the nucleosides to enhance double or triple helix formation.
  • this invention provides compositions and methods that incorporate complex structural substituents onto a polyamine chain to optimize the targeting of DNA or RNA for inhibiting replication, transcription or translation.
  • polyamines modulate various receptor or channel functions.
  • polyamines modulate the Ca 2+ -permeable glutamate receptors assembled from subunits containing a glycine residue at the RNA editing site.
  • the inward rectification of the K + inward rectifying channels is induced by blocking the outward current using cytoplasmic Mg 2+ (or intrinsic channel gating). This gating is due primarily to a block by cytoplasmic polyamines (Shyng-Si, et al. Proc. Natl. Acad. Sci. USA. 93:12014-12019, 1996).
  • polyamine analogues are useful for modulating glutamate receptors that are important in ischemia, strokes, and cardiovascular disease.
  • NMDA receptor antagonists act as anticonvulsants so that agents active at NMDA receptors are additional useful targets.
  • DFMO an ODC inhibitor
  • DFMO can cure T. brucei infection in mice and is active against African sleeping sickness in humans caused by T. brucei gambiense.
  • DFMO also has clinical utility in Pneumocystis carinii pneumonia and in infection by the coccidian protozoan parasite, Cryptosporidium.
  • DFMO acts against Acanthamoeba, Leishmania, Giardia, Plasmodia and Eimeria (Marton, L.J. et al, Annu. Rev. Pharmacol. Toxicol 35:55-91, 1995).
  • Polyamines are also essential for the growth of Hemophilus and Neisseria organisms (Cohen, S.S., A Guide to the Polyamines, Oxford University Press, NY. pp 94-121, 1998).
  • ll invention can be used to treat diseases caused by Trypanososma cruzi, T. brucei, Pneumocystis carinii, Cryptosporidium, Acanthamoeba, Leishmania, Giardia, Plasmodia, Eimeria, Hemophilus and Neisseria.
  • DFMO is an effective fungicide in the following plants: tomato plants against Verticillium wilt fungus; wheat against stem rust fungus and powdery mild fungus; bean plants against powdery mildew fungus; Macintosh apple leaves against the powdery mildew fungus; Ogle oats against leaf rust fungus; and corn against the corn rust fungus (US 4,818,770).
  • the compositions of this invention that lower polyamine levels by PAT inhibition or that have other actions on systems that utilize, or are affected by, polyamines could be useful in protecting plants against a wide range of fungi. Miscellaneous Targets
  • Polyamines may protect DNA from radiation damage (Newton, G.L. et al, Radial Res. 145:776-780, 1996). Therefore, an agent that raises polyamine levels, or that substitutes for an endogenous polyamine more effectively, may be a useful adjunct to radiotherapy. Polyamines may play an important role in controlling mammalian fertility (US 4,309,442) and maintaining embryonic growth (US 4,309,442). Other actions ascribed to polyamines include anti- diarrheal, anti-peristaltic, gastrointestinal anti-spasmodic, anti-viral, anti- retroviral, anti-psoriatic and insecticidal (US 5,656,671).
  • the present invention also has use in: (a) cleanup of toxic or radioactive metal waste that requires specific ion-binding (this can be designed into a polyamine for use in vivo or environmentally); (b) image-enhancement in medical imaging systems such as X-ray, computer-assisted tomography or magnetic resonance technologies; (c) enzyme-like catalysis that is required in asymmetric organic synthesis or resolution; (d) xenobiotic detoxification; and (e) nucleosidase activities.
  • the agent 8-chloro-cAMP (8-Cl-cAMP) (Tortora et al, Cancer Res. 57:5107-5111, 1997) is a cAMP analogue that selectively down-regulates PLA-1 , a signaling protein that is directly involved in cell proliferation and neoplastic transformation and that mediates the mitogenic effects of certain oncogenes and growth factors.
  • 8-Cl-cAMP inhibited tumor angiogenesis and secretion of growth factors of the EGF family and synergized with anti-EGF receptor antibodies in inhibiting tumor growth.
  • 8-Cl-cAMP acts by different mechanisms than do the polyamine analogues of this invention.
  • the PAT inhibitors of the present invention can be used alone, in combination with 8-Cl-cAMP, or in combination with an ODC inhibitor and/or a SAM decarboxylation inhibitor (with or without 8-Cl-cAMP). Because polyamine modulation affects chromatin structure, other agents can be used in combination with the PAT inhibitors of this invention include topoisomerase inhibitors, DNA alkylating agents and DNA intercalating agents such as doxorubicin, adriamycin, chlorozotocin, etc.
  • the present invention is directed to a series of polyamine analogues or derivatives and their use as drugs, as agricultural or as environmentally useful agents.
  • the invention defines sites and structures within these compounds that are
  • compositions of the present invention include polyamine derivatives substituted at one or more positions.
  • Disubstituted polyamines are preferably substituted at the two terminal nitrogens, but may be alternatively or additionally substituted at internal nitrogen and/or internal carbon atoms.
  • a preferred embodiment is a highly specific PAT inhibitor with pharmaceutical utility as an anti-cancer chemotherapeutic.
  • Preferred compounds with such activity include N'-dansylspermine (also termed monodansylspermine or MDS (1), N'-dansylspermidine (also termed monodansylspermidine or MDSd,
  • N'-[(N 6 -dansyl)-6-aminocaproyl]spermine (termed DACS, 4), N'-[(N 6 -dansyl)-6- aminocaproyl] spermidine (DACSd), N'-[(N 6 -5-(4-chlorobenzamidomethyl)- thiophene-2-sulfonyl)-6-aminocaproyl]spermine 5 or N'-[(N 6 -(2-dibenzofuran- sulfonyl)-6-aminocaproyl] spermine 6.
  • the latter two compounds have surprisingly high binding and inhibitory activity compared to the corresponding compounds lacking the C6 caproyl spacer between the aryl group and the polyamine.
  • DACS 4 and DACSd, and compounds 5 and 6 are preferred pharmaceutical compositions.
  • Use of alternate spacers (or linkers or couplers) and other aryl or heterocyclic "head" groups, all of which are disclosed herein, is expected to yield even more potent PAT inhibitors.
  • Preferred substituents are structures that increase binding affinity or otherwise enhance the irreversibility of binding of the compound to a polyamine binding molecule, such as the PATr, an enzyme or DNA.
  • additional substituents include the aziridine group and various other aliphatic, aromatic, mixed aliphatic-aromatic, or heterocyclic multi-ring structures.
  • Reactive moieties which, like aziridine, bind irreversibly to a PATr or another polyamine binding molecule, are also within the scope of this invention.
  • Examples of reactive groups that react with nucleophiles to form covalent bonds include chloro-, bromo- and iodoacetamides, sulfonylfluorides, esters, nitrogen mustards, etc.
  • Such reactive moieties are used for affinity labeling in a diagnostic or research context, and subserve pharmacological activity as sites within a drug that inhibit PAT or
  • the reactive group can be a reactive photoaffinity group such as an azido or benzophenone group.
  • Chemical agents for photoaffinity labeling are well-known in the art (Flemming, S.A., Tetrahedron 51 : 12479- 12520, 1995). Photoreactive compounds for cancer treatment are also known in the art.
  • composition which is a polyamine analogue or derivative that binds to a polyamine-binding site of a molecule and/or inhibits polyamine transport, which composition has the formula
  • R l is H, or is a head group selected from the group consisting of a straight or branched C,. 10 aliphatic, alicyclic, single or multring aromatic, single or multiring aryl subsituted aliphatic, aliphatic-substituted single or multiring aromatic, a single or multiring heterocyclic, a single or multiring heterocyclic-substituted aliphatic and an aliphatic-substituted aromatic;
  • R 2 is a polyamine; and X is CO, NHCO, NHCS, or SO 2
  • R 2 has the formula NH(CH 2 ) n NH(CH 2 ) p NH(CH 2 ) q NHR 3 wherein
  • R 3 is H; C,. ]0 alkyl; C,. I0 alkenyl; C,. 10 alkynyl; alicyclic; aryl; aryl- substituted alkyl, alkenyl or alkynyl; alkyl-, alkenyl-, or alkynyl- substituted aryl; gauanidino; heterocyclic; heterocyclic-substituted alkyl, alkenyl or alkynyl; and alkyl-, alkenyl-, or alkynyl- substituted heterocyclic.
  • composition may further comprise, linked between X and R 2 , a linker L and an additional group y, such that said composition has the formula: R,-X-L-Y-R 2 wherein, L is a C,. ]0 alkyl, C,. 10 alkenyl, C,. 10 alkynyl, alicyclic, or heterocyclic; X is CO, SO 2 , NHCO or NHCS; and
  • Y is CONH, SO 2 NH, NHCO, NHCONH, NHCSNH, NHSO 2 , SO 2 , O, or S.
  • R can havethe formula:
  • R has the formula:
  • R 4 and R 5 are, independently, H, OH, halogen, NO 2 , NH 2 , NH(CH) n CH 3 , N((CH) n CH 3 ) 2 , CN, (CH) n CH 3 , O(CH) n CH 3 , S(CH 2 ) n CH 3 , NCO(CH 2 ) n CH 3,
  • R has the formula:
  • Q is CONH, SO 2 NH, NHCO, NHCONH, NHCSNH, NHSO 2 , SO 2 , O, or S.
  • R may have the formula:
  • r and s vary independently and are 0 to 6;
  • R 4 , R 5 , R, and R 7 are, independently, H, OH, NO 2 , NH 2 , NH(CH) n CH 3 ,
  • N((CH) n CH 3 ) 2 , CN, (CH) n CH 3 , O(CH) n CH 3 , S(CH 2 ) n CH 3 , NCO(CH 2 ) n CH 3, O(CF 2 ) n CF 3 , or CO-O(CH) n CH 3 where n 0 to 10;
  • Q is CONH, SO 2 NH, NHCO, NHCONH, NHCSNH, NHSO 2 , SO 2 , O, or
  • R may be selected from the group consisting of naphthalene, phenanthrene, anthracene, pyrene, dibenzofuran, acridine, 2,1,3-benzothiodiazole, quinoline, isoquinoline, benzofuran, indole, carbazole, fluorene, 1,3-benzodiazine, phenazine, phenoxazine, phenothiazine, adamantane, camphor, pipiridine, alkylpiperazine, morpholine, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, thiophene, furan, pyrrole, alkyl- 1,2-diazole, alkylimidazole, alkyl- 1H-1, 2,3 -triazol, alkyl-lHl,2,3,
  • R may also be a D- or L-amino acid.
  • composition where R has a formula selected from the group consisting of
  • R 12 and R ]3 are H, naphthalene, phenanthrene, anthracene, pyrene, dibenzofuran, acridine, 2,1,3-benzothiodiazole, quinoline, isoquinoline, benzofuran, indole, carbazole, fluorene, 1,3-benzodiazine, phenazine, phenoxazine, phenothiazine, adamantane, camphor, pipiridine, alkylpiperazine, morpholine, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, thiophene, furan, pyrrole, alkyl- 1,2-diazole, alkylimidazole, alkyl- 1H-1, 2,3 -triazol, alkyl- 1 HI, 2,3, 4-tetrazol
  • Y, and Z are CONH, SO 2 NH, NHCO, NHCONH, NHCSNH, NHSO 2 * NHSO 2 , SO 2 -NHSO 2 , SO 2 , O, S, COO or when R, is of formula (A) or (B), Y, represents a bond between a C or N atom of R 12 and a C or N atom of R 13 and Z, represents a bond between a C or N atom of R 13 and a C or N atom ofR 14 ; or when R, is of formula (C) or Y, represents a bond between the C and a C or N atom of R 13 and Z, represents a bond between the C and a C or N atom of R, 4 ; or when R, is of formula (D) Y, represents a bond between a C or N atom of R I2 and a C or N atom of R 14 and Z, represents a bond between a C or N atom of R 13 and a C or N atom
  • R 3 is H; C,. 10 alkyl; C,. 10 alkenyl; C 0 alkynyl; alicyclic; aryl; aryl- substituted alkyl, alkenyl or alkynyl; alkyl-, alkenyl-, or alkynyl- substituted aryl; gauanidino or heterocyclic; and
  • (c) Z isCH 3 , CH 2 CH 3 or cyclopropyl.
  • R 2 has the formula:
  • R 2 is preferably seleccted from the group consisting of N'-acetylspermine, N'-acetylspermidine, N 8 -acetylspermidine,
  • compositions are polyamine analogues selected from the group consisting of compounds designated herein 3, 4, 5, 6, 13, 14, 29, 40, 43, 44, 45, 57, 58, 56, 66, 67, 72, 76, 84, 88, 89, 95 and 96, most preferably, compound 4, 5, 6, 43, 65, 66, 84, 89, 95 or 96.
  • R, or R 3 may be bonded at one or more sites to a reactive moiety that is capable of forming covalent bonds with a nucleophilic site on a target molecule, such as a protein or a nucleic acid, preferably a cellular receptor or other cell surface molecule.
  • a target molecule such as a protein or a nucleic acid, preferably a cellular receptor or other cell surface molecule.
  • Such composition permit esssentially irreversible binding that is advantageous in both diagnostic and therapeutic uses.
  • the present invention is also directed to a pharmaceutical composition useful for treating a disease or condition in which the inhibition of polyamine transport is desirable, comprising a composition as described above and a
  • the pharmaceutical composition may further include a an inhibitor of polyamine synthesis; preferably DFMO.
  • Other combinations include the above pharmaceutical composition and one or more additional agents known to be useful for treating said disease or condition
  • This invention also provides amethod for treating a disease or a condition in a subject associated with undesired cell proliferation and/or which is treatable by inhibition of polyamine transport, comprising adminstering to said subject an effective amount of a pharmaceutical compsition as described above.
  • the undesired cell proliferation may be associated with proliferation of cells of the immune system, cell of the vascular neontima, tumor cells or with undesired angiogenesis.
  • Preferred diseases to be treated as above include cancer or post-angioplasty injury.
  • the present invention is directed to a series of polyamine analogues useful in an improved assay of polyamine uptake into the cell or polyamine binding to specific ligands.
  • the invention identifies elements that are key for polyamine binding to membrane proteins such as the PATr (PATr), and to soluble proteins, and which can be monitored through this technique.
  • PATr PATr
  • Disubstituted polyamines preferably having a reactive group at one end, may also be employed as assay or biochemical probes.
  • a preferred assay method employs a monosubstituted polyamine probe having a moiety that serves as a detectable label (a "reporter”), preferably a fluorophore, most preferably the dansyl group, or another substituent that can be detected through a variety of means, including by ELISA.
  • a detectable label preferably a fluorophore, most preferably the dansyl group, or another substituent that can be detected through a variety of means, including by ELISA.
  • a preferred assay method employs a polyamine or analogue immobilized to a solid support.
  • Additional substituents which may be present on the polyamine core are structures which increase binding affinity, or otherwise enhance the irreversibility of binding of the compound to a polyamine binding molecule, such as a PATr, an enzyme or DNA.
  • additional substituents include the aziridine group and various other aliphatic, aromatic or heterocyclic multi-ring structures.
  • a reactive moiety, which, like aziridine, can bind irreversibly to a PATr or another polyamine binding molecule is also contemplated.
  • groups which react with nucleophiles to form covalent bonds include chloro-, bromo- and iodoacetamides, sulfonylfluorides, esters, nitrogen mustards, etc.
  • Such reactive moieties are used for affinity labeling in a diagnostic or research context, and subserve pharmacological activity as parts of drugs that inhibit PAT or polyamine synthesis.
  • the reactive group can also be a reactive photoaffinity group such as an azido- and benzophenone group.
  • the present invention includes a high throughput screening assay which allows processing of high numbers of potential target compounds that are being screened for activity as inhibitors, preferably competitive inhibitors, of PAT.
  • the method is suited to the 96 well microplate format for use with robotic sample and plate handling systems known in the art.
  • the means for detecting transport are a function of the detectable label used. Fluorescence and chemiluminescence are the methods of choice.
  • the present invention also encompasses a unique pharmacophore for a polyamine binding site that can be used to isolate a polyamine binding target or to assay the conformational state of a selected target by its binding.
  • an enzymatic assay which permits amplification of the signal, wherein biotin or some other "reporter” is conjugated to the polyamine as the “detectable label,” and is detected by allowing the binding of streptavidin conjugated to an enzyme, followed by generation of a colored product of the enzyme from a chromogenic substrate.
  • a composition in which both biotin and a "hapten" group recognized by an antibody are coupled to a polyamine. In another composition the hapten is coupled directly to the enzyme.
  • the diagnostic or assay composition is designed to be used in an assay of polyamine transport or polyamine binding, and comprises a polyamine analogue as set forth above, that is detectably labeled and/or includes at least one reporter group capable of detection.
  • the analogue comprises a linker group L between the reporter group and said polyamine.
  • a preferred reporter group is a fluorophore, a chromophore or a luminescer, most preferably dansyl or biotinyl; the polyamine is preferably spermine, spermidine or putrescine. Most preferred for this utilityis monodansylspermine or DACS.
  • the diagnostic composition may also include as a single, or as one of several reporter groups, a hapten recognized by an antibody.
  • the above diagnostic compositions may have the polyamine analogue bound to an enzyme.
  • the polyamine analogue is immoblized to a solid support.
  • An assay method provided herein for detecting polyamine transport comprises
  • the assay comprises (a) incubating molecules or cells having a polyamine binding site or cells having a polyamine transporter with (i) a polyamine analogue of any of claims 26-33, (ii) with and without said unknown compound; (b) measuring the quantity of said reporter bound to said cells or molecules or internalized in said cells; and (c) comparing the amount of reporter bound or internalized in the presence of said unknown compound to the amount of reporter bound or internalized in the absence of said unknown compound, wherein a reduction in the amount of said reporter detected is measure of the binding or transport of said unknown compound.
  • the methods of the present invention include high throughput solid phase synthesis of a polyamine library.
  • This library includes the attributes of a solid phase support, a cleavable linker that attaches the molecule to the support, the addition of extenders that are a series of protected aldehydes, amino acids, etc.,
  • Figure 1 shows the structure and activity relationships (SAR) between spermidine, MDS and DACS.
  • K j values are the inhibitory constants obtained in a PAT inhibition assay.
  • Figure 2 (sheets 2/1 to 2/10) is a tabular representation of a large number of chemical structures 3-98 that were tested for their effects on cell growth.
  • R an index of growth inhibitory activity, is the ratio of the growth of cells in the presence of the test compound to the growth in the presence of the compound plus DFMO.
  • the K j5 (inhibition constant) reflects a compound's inhibition of PAT in cell culture. These biological effects provide a basis for SAR analysis.
  • Figure 3 shows synthetic routes to N'-substituted polyamine analogues
  • Figure 4 is a scheme of the synthesis of N-(l-anthracenyl)-N'-(N'- spermidyl)urea 9
  • Figure 5 is a scheme of the synthesis of N'-(l-pyrenylsulfonyl)spermine 15
  • Figure 6 shows a scheme of the synthesis of N'-((l-carbonyl)-4-(l- pyrenyl)butane)spermine 7
  • Figure 7 shows a scheme of the synthesis of the synthesis of N ! -dansyl- spermine 3 (MDS).
  • Figures 8 and 9 each show a different synthetic scheme for the synthesis of
  • Figure 10 shows four classes (111-114) of conformationally restricted polyamine analogues, and at the bottom, a stereochemically defined, internally cyclic polyamine analogues (116).
  • Figure 1 1 is a synthetic scheme wherein free primary amino groups are blocked by N-acylation (44) and N-alkylation (77), thereby reducing potential metabolic degradation of the derivatized PAT inhibitors.
  • Figure 12 is a synthetic scheme for bis ⁇ -gem-dimethylpolyamine analogues 121.
  • Figure 13 is a synthetic scheme for internally substituted polyamine analogues containing cyclopropyl groups (122-126)
  • Figure 14 is a synthetic scheme for internally substituted polyamine analogues containing a C-C branch (127-134)
  • Figure 15 shows examples of spacers or linkers for use with multiring head group (135-139).
  • Figure 16 shows a series of compounds (140-147) containing multiple ring head groups.
  • Figure 17 is a graph showing the effects of DACS on growth of MDA breast cancer cells with and without DFMO.
  • Figure 18 is a graph showing the effects of headless polyamine analogues on growth of PC-3 prostate cancer cells with and without DFMO.
  • Figure 19 lists chiral carbon-substituted amino acid linker groups.
  • Figure 20 is a scheme of the synthesis of N'-(aziridinyl)-N 12 -[(N 6 -dansyl)- 6-aminocaproyl]spermine 157.
  • Figure 21 is a scheme of the synthesis of a di-substituted aziridinyl polyamine analogue 160.
  • Figure 22 is a graph showing inhibition of the growth of MDA-MB-231 cells, by DACS in the presence ( ⁇ ) or absence ( ⁇ ) of the polyamine synthesis inhibitor DFMO. See also, Figure 2/1-2/10 for the effects of a large number of polyamine analogues on PAT and tumor cell growth. Cells were plated in the presence of varying concentration of DACS with and without 1 mM DFMO. Cells numbers (expressed as % of controls) were determined after 6 days as above.
  • Figure 23 is graph showing inhibition of cell growth in the presence of
  • Figure 24 is a graph showing the inhibition of growth of PC-3 prostate cancer cells by the combination of DACS and DFMO. See description of Figure 22 for conditions and details.
  • Figure 25 shows a group of chemical structures (161-165) including three known psychoactive compounds trifluoperazine 163, thorazine 164 and imipramine 165. Compounds 161, 162 and 165 inhibited polyamine transport.
  • Figure 26 is a graph showing the inhibition of spermidine/spermine acetyltransferase (SSAT) enzymatic activity by DACS.
  • Figure 27 is a graph showing a comparison of the kinetics of uptake of N'-monodansyl spermine (MDS) with the uptake of radiolabeled spermidine.
  • MDS concentrations were as follows: ⁇ 0 ⁇ l ⁇ M ⁇ 0.3 ⁇ M X
  • Figure 28 is a graph showing detection of MDS in the absence of DFMO by fluorescence in A 172 glioblastoma cells.
  • Figures 29 and 30 describe the synthesis of a biotin modified polyamines
  • N i [(N 6 -(biotinyl)-6-aminocaproyl)]spermine and N 1 -(biotinyl)spermine.
  • Figure 31 is a schematic illustration showing the possible sites for modifying a polyamine to create an "immobilization handle” and a “reporter handle” combination.
  • Figure 32 is a graph showing the detection of Nl -dansylspermine and
  • Figure 33 is a general scheme that brings together the three major components of the present compositions in a synthetic cycle for generating polyamine derivatives.
  • Figure 34 outlines synthesis of an activated tert-alkoxycarbonyl MeO-
  • Figure 35 shows the production of these extenders from either commercially available amino alcohols or the chiral amino acid precursor pool.
  • Figure 36 shows the next step in the synthetic cycle: reductive amination with NaBH 3 CN is used to initially extend the backbone followed by an additional
  • Figure 37 shows the final steps, including the final capping and the acid- mediated cleavage of the product from the polymeric support as the trifiuoroacetate salt of the desired analogue.
  • Figure 38 shows "modifications" of polyamine analogues as they are extended with aldehydic nucleoside terminators. Each amino group can be dressed individually and specifically with any of the four ribonucleosides or 2'-deoxyribonucleosides.
  • Figure 39 shows an example of a solid support with alternative linking groups used for solid phase synthesis of polyamine libraries. 3,4-dihydro-2H- pyran-2-yl-methoxymethyl polystyrene is shown.
  • Figure 40 shows various linkers used in a multipin method of dimensionally stable polypropylene/polyethylene pins to which a graft polymer is covalently linked.
  • the Rink amide linker is shown as structure 23a coupled to the pin.
  • Figure 41 shows a compound that is synthesized using a solid support and the synthetic approach described for Figures 4 and 5.
  • Compound 31a is synthesized using the blocked 3-aminopropanal 27a as the first extender, benzaldehyde 28a as the first terminator, the blocked methioninal 29a as the second extender and acetone as the final terminator.
  • the present inventors have designed novel compounds for therapeutic uses and have devised tests using such compounds as probes for measuring PAT and polyamine binding in an efficient, high throughput assay. Using the novel methods, they have screened for and discovered compounds with high affinity for the PATr that inhibit uptake, both competitively and non-competitively. Such compounds are useful as drugs in a number of diseases, particularly cancer. They can also be used as a component of novel drug combinations with, for example, a
  • polyamine synthesis inhibitor such as DFMO (which inhibits ornithine decarboxylase) or with other agents.
  • the compounds of the present invention are also useful in other diseases or conditions in which polyamines play a role as described above, and have agricultural and environmental uses.
  • the inventors found that various chemical groups can be attached to a polyamine to give it advantageous properties as an inhibitor of PAT or as a probe in an assay of PAT and for drug screening. Such chemical modification does not destroy the effective binding and, in fact, enhances the affinity of the derivatized polyamine for the PATr. Hence, these compounds are useful for discovery of inhibitors of polyamine uptake.
  • polyamine is intended to mean putrescine, spermine or spermidine, as well as longer linear polyamines, branched polyamines, and the like, which may have between 2 and about 10 nitrogens. Also included in this definition are polyamine derivatives or analogues comprising a basic polyamine chain with any of a number of functional groups bound to a C atom or a terminal or internal N atom. A polyamine derivative may include a terminal linker or spacer group between the polyamine core and a derivatizing function. A "head group” is defined as a moiety bonded either directly to the polyamine or attached to a linker that is bonded to the polyamine.
  • a head group may be a fluorescent moiety, which also serves as a "reporter.”
  • An "inhibitor” moiety or group is a chemical group derivatizing a polyamine that (1) causes the derivative to bind to the PATr with higher affinity than does a native polyamine and/or (2) by other means blocks the uptake of a polyamine (or a probe of this invention) into a cell or a subcellular PATr preparation.
  • the inventors disclose herein compounds that efficiently inhibit PAT in MDA-MB-231 human breast carcinoma cell and other cells.
  • a reporter moiety is a chemical moiety forming part of a probe which renders the probe detectable (either directly or, for example, through enzymatic enhancement) and hence permits the determination of the activity of the PATr to which the probe binds.
  • a reporter is detectable either because it itself emits a detectable signal, or by virtue of its affinity for a reporter-specific partner which is detectable or becomes so by binding to, or otherwise reacting with, the reporter.
  • polyamine analogue is immobilized to a solid support which enables removal of the analogue and any interacting/binding molecules from a complex mixture.
  • the PAT inhibitors were developed by modification of the natural substrate of the transporter, spermidine.
  • the present inventors discovered that introduction of a 3-amidopropyl group to the diaminobutyl part of spermidine produced a significantly better transport inhibitor as shown in Figure 1.
  • the optimal amido or sulfonamide substituent was found to be a medium sized aromatic group, leading to the invention of N'-dansylspermine (MDS) as both a transport inhibitor and a transport assay reporter molecule.
  • MDS has increased binding affinity to cells compared to spermidine and N'-acetylspermine.
  • DACS N'-[(N 6 -dansyl)-6- aminocaproyl] spermine
  • a series of inhibitors was made by direct reaction of a polyamine with a sulfonyl chloride, acyl, isocyanate, isothiocyanate, alkyl chloride or an
  • N-hydroxysuccinamide-activated carboxy ester as described in Figure 3 and in Examples I-IV. Different head groups, linkages and polyamines were combined. Many of the Figures show spermine as a nonlimiting example of the polyamine core of the molecule. The polyamine core can be varied as defined above. The synthesis of N 1 -
  • N-(l-anthracenyl)-N'-(N'-spermidyl)urea 9 from 1 -aminoanthracene and spermine ( Figure 4) is described in more detail in Example IV.
  • Urea derivatives can also be synthesized using substituted isocyanates. For example, 1- aminoanthracene is first activated with p-nitrophenyl chloroformate to form the urethane which is reacted with spermine to yield a substituted urea 9.
  • PAT inhibitors of this group have spermine as the polyamine core and include a head group such as pyrenyl (see Figure 5; Example II (15)), 5-(4- chlorobenzamidomethyl)thiophenenyl (13) or dansyl (3) ( Figure 7; Example I).
  • Inhibitors of this type typically have K, values of approximately 100 nM and R values in the MDA growth assay of >1.
  • Example I the polyamine in CH 2 C1 2 solvent was treated dropwise to a solution of the acid chloride in the same solvent. This gave a statistical mixture of the unsubstituted, monosubstituted and disubstituted polyamine derivatives, which is advantageous because purification by the methods described herein resulted in pure mono- and di- substituted derivatives.
  • Each analogue was then tested in the biological assays (PAT inhibition and cell growth inhibition). It was sometimes an advantage to produce an individual mono-substituted derivative using a mono- protected polyamine intermediate. Large-scale (> 5 grams) production of the analogues was accomplished in this fashion because removal of side products was greatly facilitated.
  • the preferred mono-protected polyamine intermediates were the N'-tBoc derivatives produced according to Blagbrough et al, ⁇ Tetrahedron Lett. 35:2057-
  • the product can be separated on a weak cation exchanger such as BioRad ® 70, with a NH 4 OH gradient.
  • a weak cation exchanger such as BioRad ® 70
  • NH 4 OH gradient a weak cation exchanger
  • ⁇ e.g., protein has been to synthesize conformationally or stereochemically defined analogues of a binding molecule.
  • a molecule By significantly reducing the number of possible rotomers or conformations a molecule can adopt, one can attain increased binding to the desired site. Since the molecule no longer has to search the entire "conformational space," its energy of interaction with the target increases many times.
  • Ganem replaced the butyl portion of spermine with 2-butene and 2-butyne diamino derivatives (Ganem, B., J. Org. Chem. 1987, 52, 5044-5046). Rajeev, K.G. et al,
  • the present inventors extended this work by producing the other analogues shown in Figure 10. These analogues are synthesized using variations of known methods.
  • the primary amines are protected as N-tBoc derivatives for the analogues 111 and 113. Acid deprotection then gives the desired products.
  • the derivative 112, where x - 1 was also synthesized Ganem.
  • the natural polyamines including putrescine, spermidine and spermine, are incorporated into the compositions of this invention by coupling them to the various "head” and "linker” groups.
  • Other naturally occurring polyamines that can be employed similarly include: N'-acetylspermine, N'-acetylspermidine, N 8 - acetylspermidine, N'-guanidinospermine, cadaverine, aminopropylcadaverine, homospermidine, caldine (norspermidine), 7-hydroxyspermidine, thermine
  • thermospermine 5 ⁇ (norspermine), thermospermine, canavalmine, aminopropylhomospermidine, N, N'-bis(3-aminopropyl)cadaverine, aminopentylnorspermidine, N 4 -aminopropylnorspermidine, N 4 -aminopropylspermidine, caldopentamine, homocaldopentamine, N 4 -bis(aminopropyl)norspermidine, thermopentamine, N 4 -bis(aminopropyl)spermidine, caldohexamine, homothermohexamine and homocaldohexamine.
  • the metabolic stability in vivo of monosubstituted polyamine analogues is increased by modifying these compounds to resist enzymatic degradation. For example, substitution of the terminal primary amine group with an alkyl group would achieve this by preventing oxidative metabolism.
  • This invention also includes compounds with alkylated secondary amino groups. N-alkylation of the amide nitrogens slows down proteolytic degradation.
  • the terminal free primary amino group can be blocked by N-alkylation (Bergeron, R.J. et al, J. Med. Chem. 37:3464-347, 1994) as illustrated in Figure
  • methyl groups can be introduced ⁇ to the terminal amino groups (121) of spermine (Lakanen, J. R. et al, J. Med. Chem. 35:724-734, 1992).
  • the 1,12-dimethylspermine analogue 121 was very resistant to normal metabolic degradation. This compound is easily coupled to a linker and head group as shown in Figure 12 (compounds 66, 18, 121).
  • Ganem, B., J. Org. Chem. 1986, 51, 4856-4861 synthesized bis ⁇ -gem-dimethylpolyamine analogues. The present inventors have extended upon these two reports and synthesized the bis- cyclopropylamine analogues by the route described below. See Figure 13.
  • Chem. 1997, 62, 1584-1585 produced the fully protected bis-cyclopropylamino analogue of spermine. Catalytic hydrogenation yields a fully deprotected polyamine.
  • cyclopropyl-substituted polyamine analogues can be produced in an analogous manner to that shown in Figure 13. Other analogues produced are shown at the bottom of Figure 13. These cyclopropyl polyamine analogues are activated by cellular enzymes to become alkylating agents.
  • MB-231 cell PATr of 2100, 41, 18 nM, respectively.
  • Detectably labeled polyamine derivatives can be synthesized using radiolabeled 14 C-spermine or other radiolabeled polyamine as starting material.
  • the resulting acid chloride can then be reacted with various nucleophilic reagents to produce carboxy-substituted polyamine analogues following removal of the tBoc group. These analogues can then be coupled to the reagents that donate the linker and/or head group. Alternatively, the carboxy intermediate can be reduced to an intermediate that is used to synthesize numerous analogues.
  • Such analogues are of interest in the present invention as alkylating agents ⁇ e.g., internal aziridine spermine derivatives) or as enzyme-activated irreversible inhibitors of enzymes involved in polyamine biosynthesis, utilization and degradation ⁇ e.g., spermine synthase, deoxyhypusine synthase, polyamine oxidase) as shown in Figure 14 (compounds 130-134). Any enzyme that acts on alkylating agents ⁇ e.g., internal aziridine spermine derivatives) or as enzyme-activated irreversible inhibitors of enzymes involved in polyamine biosynthesis, utilization and degradation ⁇ e.g., spermine synthase, deoxyhypusine synthase, polyamine oxidase) as shown in Figure 14 (compounds 130-134). Any enzyme that acts on
  • a number of coupling chemistries can be used to combine the "head” group and the linker moiety.
  • Types of "head” groups are disclosed below as are additional groups that can be substituted onto these head groups.
  • halogen cyclohexyl ethoxyl propyl ester methyl cycloheptyl propoxyl isopropyl ester ethyl cyclooctyl thio cyano propyl cyclononyl methylthio isocyanato isopropyl cyclodecyl ethylthio trifluoromethyl butyl hexyl propylthio trichloromethyl isobutyl 2-hexyl butylthio tribromomethyl tert-butyl 3 -hexyl isopropylthio azido pentyl allyl nitro Acetoxy
  • Aromatic groups include phenyl naphthyl, 1-, 2-, or 3-biphenyl, indenyl, acenaphthylenyl, anthracenyl, phenanthrenyl, phenalenyl, triphenylenyl pyrenyl, diphenylmethylenyl, etc.
  • Heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, biphenyl, furanyl, pyrrolyl, 1 ,2-diazolyl, imidazolyl, 1 H, 1,2,3 - triazolyl, lH-l,2,3,4-tetrazolyl, thiazolyl, oxazolyl, 1,3,4-thiadiazolyl, pyridinyl, pyrimidyl, 1 ,2-diazinyl, 1 ,4-diazinyl, 1,3,5-trizinyl, dibenzofuranyl, acridinyl, 2,1,3-benzothiadiazole, isoquinolinyl, quinolinyl, benzufuranyl, isobenzofuranyl, 1,3-benzodiazinyl, phenazinyl, phenoxazinyl, phenothiazinyl, pyran
  • This class includes straight-chain, branched and cyclic hydrocarbons attached to the linker.
  • the group includes C 2 . 10 alkanes; C 3 . ]0 alkenes containing 1 to 3 unsaturations; C 3 ., 0 alkynes containing 1 to 3 unsaturations; branched C 3 . 10 alkanes, alkenes and alkynes; polycyclic aliphatic hydrocarbons and steroid-like ring systems that include C 3 . 8 cycloalkyl, adamantyl, camphoryl, cholesteryl, etc.
  • intercalating agents amenable to this use are acridine, 9-aminoacridine, proflavine, actinomycin D, daunorubicin, doxorubicin, nogalamycin, menogaril, ellipticine, BD-40, amsacrine, acodazole, 2-pheylquinoline carboxamide, crisnatol, nitracrine, pyrazoloacridine, mitonoafide, ametantrone, mitoxantrone, oxanthrazole, bisantrene, echinomycin.
  • the following biochemicals are candidates for coupling to polyamines for producing a selective pharmaceutical agent: steroids, prostaglandins, phospholipids; enzyme cofactors including nucleotide containing molecules such as NADH, AcetylCoA, AdoMet, flavin, tryptophantryptophyl quinone (TTQ), etc.
  • An additional series of head groups comprises polyamines conjugated to polyethylene glycol (PEG) or O-methylated PEG (abbreviated MeOPEG) polymers of various sizes.
  • PEG polyethylene glycol
  • MeOPEG O-methylated PEG
  • Head groups can vary from simple alkyl substitutions to multi-ring and multi-single-ring substitutions. Some of the structural variations are schematically represented in Figure 15.
  • Spacers X, Y and Z are defined as bonds or straight chain groups that attach different ring structures in a multiple ring head group. In some cases the spacers function as direct C-C or C- N attachments. Conventional spacers known in the art are similar to the linkers described herein. Known chemistries are used for covalent attachment of a ring structure in a head group with a spacer, for example, the formation of amide, sulfonamide, ether, thioether, ester, -C-C- and -C-N- and -N-N- bonds. Rnch R 2 and R 3 are typically alicyclic, aromatic, or heterocyclic rings when substituted in multi-ring head groups. These ring structures individually can also be substituted. Some of the multi-ring head group types described above are available from commercial sources, and examples are shown as structures 140 to 147 in Figure 16. Alternatively, these or similar compounds are readily synthesized. Linker Group
  • the linker portion of the compound can be represented by a general structure with an amino group at one end and an acid group on the other.
  • One group of linkers contains diamino groups that are bonded via a urea linkage to the polyamine and via an amide, urea or sulfonamide linkage to the head group.
  • the head group can also be bonded through other couplings such as ether, thioether and C-C bonds.
  • the schematic structure shown above shows the function of the linker moiety connecting the head group to the polyamine and possessing a desired length and combination of steric, conformational and hydrophobic properties. Also shown are the possible combination of coupling methods. Each coupling method can be used in combination with any of the three methods in Figure 3 at the other position to result in a wide array of desired properties.
  • the linker group can have a range of properties that are reflected by the number of variations discussed below. Changes in the linker structure will be affect the properties of the whole polyamine analogue such as hydrophobicity, hydrophilicity, distance between head and polyamine portions, steric arrangement of head and polyamine portions, conformational properties, solubility and electronic properties.
  • n 1 to 12
  • linker length had dramatic effects on the PAT inhibitory activity and the cell growth inhibitory activity.
  • a low K j is optimal for C 6 linkers in the presence of an aromatic head group.
  • differences in growth or transport inhibitory activities have not been dramatic.
  • "headless” compounds have KjS in the order of about 25nM but have more attenuated inhibitory effects cell growth (breast cancer cell line) most likely due to their ability to actually be transported.
  • the prostate cancer cell line is more powerfully inhibited by these "headless” inhibitors as shown in Figure 18 and Example XI.
  • the C3-headless compound had dramatic effects on cell growth.
  • ⁇ -amino acid analogues known in the art can be used to form polyamine adducts. These are very easily inco ⁇ orated into the present invention through the synthetic sequences described in Figures 8 and 9.
  • Several key examples are; t-butylglycine, ornithine, -aminoisobutyric acid, 2- aminobutyric acid, ⁇ -aminosuberic acid, 4-chlorophenylalanine, citrulline, ⁇ - cyclohexylalanine, 3, 4-dehydroproline, 3, 5-diiodotyrosine, homocitrulline, homoserine, hydroxyproline, ⁇ -hydroxvaline, 4-nitrophenylalanine, norleucine, norvaline, phenylglycine, pyroglutamine, ⁇ -(2-thienyl)alanine, etc.
  • Several important ⁇ -amino acids are easily inco ⁇ orated into the present invention through the chemistry discussed above
  • headless derivatives containing a polyamine and linker without a head group were synthesized and tested. These derivatives are made by reacting the active ester (p-nitrophenyl or
  • N-hydroxylsuccinimide of the N-tBoc amino acid with the polyamine of interest.
  • the resulting N-tBoc protected derivatives are then purified by cation-exchange chromatography over BioRex 70 (NH 4 form) resin using a linear gradient from 0 to 2N NH 4 OH.
  • the tBoc group can then be cleaved by acid treatment. Both the tBoc and acid deprotected derivatives can be tested for biological activity.
  • the full series of amino acids discussed above, together with other derivatives have been synthesized. A more detailed discussion of the synthesis of N'-[6- aminocaproylspermine] appears in Example XIV.
  • Aziridines Polyamines substituted with fluorophores and other bulky end group were found to have the intrinsic property of high avidity binding to the PATr. This suggested that, in addition to utility as a diagnostic or research tool, they are useful as therapeutic agents for treating diseases or conditions wherein it is
  • the polyamine core is substituted with the aziridinyl group.
  • the embodiment shown in Figure 20 has a second substituent (a fluorophore such as dansyl or another bulky group).
  • Aziridinyl-substituted polyamines react with nucleophilic groups in target binding complexes (receptors, transporters, enzymes and nucleic acids). In addition they can be exploited to bind other reactive moieties to polyamines.
  • target binding complexes receptors, transporters, enzymes and nucleic acids.
  • These mono- and di-substituted polyamine analogues are useful as drugs because of their inhibition of (a) the PATr, (b) polyamine synthesis and (c) reactions that use nucleic acids as substrates.
  • a reactive group other than aziridine is introduced into a polyamine already substituted with a head group and a linker. This reactive group allows the labeled polyamine to bind covalently to an appropriate nucleophilic site on a polyamine-binding target molecule such as the PATr.
  • Disubstituted polyamines are synthesized by using the appropriate amine protecting groups on the polyamines. Reagents for the step wise fuctionalization of spermine are known (Bergeron, R.J. et al, J. Org. Chem. 53: 3108-3111
  • Figure 20 shows the synthesis of the spermine derivative
  • any other polyamine derivative can be produced using an appropriately protected polyamine precursor, coupling to the linker/head group moiety and reductive amination with 3-aziridinepropanal. Removal of the protecting group(s) then gives the desired, reactive polyamine derivative.
  • An additional example of this approach, illustrating the chemical flexibility it permits, is shown in the Figure 21 (158-160).
  • Felschow et al. attached an azidobenzoic acid moiety to spermine and examined the interaction of the resulting adduct with cell surface proteins (Felschow, DM et al. Biochem. J. 328, 889-895, 1997; Felschow, DM et al, J. Biol Chem. 270:28705-28711, 1995). Since their photoprobe had an
  • PATr protein(s) and would also find use as an irreversible, photoactivatable drug molecule.
  • Polyamine analogues with photoactivatible head groups are made using p-nitrophenyl 3-diazopyruvate, a reagent for introduction of a photoactivatable 3-diazopyruvate group to an aliphatic amine.
  • This agent is also available from Molecular Probes, Inc.
  • the desired derivative is made by reacting this reagent with the free amino, p-nitrophenyl activated linker precursor, purifying the linker/head group intermediate, and reacting it with the polyamine.
  • Reporter Molecules for PAT and other Polyamine-binding Proteins
  • Various moieties can be attached to polyamines to produce novel inhibitors of PAT with utility as probes in a PAT assay and for drug screening. Such chemical modifications do not destroy effective binding and, in fact, enhance the affinity of the derivatized polyamine for the PATr. Such compounds are thus useful for measuring polyamine uptake and, more importantly, in high throughput screening assays to discover therapeutically useful inhibitors of this uptake.
  • polyamine analogue is immobilized to a solid support which enables removal of the analogue and any interacting/binding molecules from a complex mixture.
  • polyamine binding protein Because a number of polyamine binding protein are targets for therapeutic intervention, an improved assay for polyamine binding or uptake is desirable. Small changes in polyamine structure can have drastic effects on activity. Reporter molecules are polyamine analogues that are conveniently detectable while maintaining their binding activity to the PATr. For optimal binding to the
  • N 1 -substituted polyamines are competitive inhibitors for the PATr and for several other polyamine binding proteins such as the external sensing Ca +2 receptor and the NMDA receptor.
  • the inventors have therefore focused on producing suitable reporter- bound polyamines that maintain a reasonable range of detectability and that bind competitively with spermine.
  • Figure 1 illustrates a synthetic scheme for various N'-linked polyamine analogues.
  • the Head/ Reporter group structural diversity is very large and includes most organic groups that can be covalently bonded to an amine.
  • M ⁇ l described below are but several examples of the head groups of the present invention and are not intended to be limiting.
  • the dansyl fluorescent group can be substituted by any of a large number of fluorophores, for example, as disclosed in Haugland, Handbook of Fluorescent
  • dansyl-polyamine-like molecules are known and commercially available, including: didansylcadaverine, monodansylcadaverine, didansylputrescine, MDS and tridansylspemidine (Sigma Chemical Company, St. Louis, MO).
  • didansylcadaverine monodansylcadaverine
  • didansylputrescine MDS
  • tridansylspemidine Sigma Chemical Company, St. Louis, MO.
  • the syntheses of monodansylputrescine Ros, D. E. et al, J.
  • a fluorescent reagent is selected based on its ability to react readily with an amino function of a polyamine such as spermine.
  • fluorescent probes include the BodipyTM (4,4-difluoro-4-bora-3a,4a-diaz-s- indacene) fluorophores which span the visible spectrum (US 4,774,339; US 5,187,288; US 5,248,782; US 5,274,113; US 5,433,896; US 5,451,663).
  • the preferred member of this group is 4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s- indacene-3-propionic acid.
  • Preferred fluorophores for derivatizing polyamines according to this invention are those which are excited by ultraviolet light.
  • Suitable fluorescent materials include Cascade BlueTM, coumarin derivatives, naphthalenes (of which dansyl chloride as exemplified herein is a member), pyrenes and pyridyloxazole derivatives, Texas redTM, BodipyTM, erythrosin, eosin, 7-nitrobenz-2-oxa-l,3-diazole (NBD), pyrenes, anthracenes, acridines, fluorescent phycobiliproteins and their conjugates and fluoresceinated microbeads.
  • Use of certain fluors such as the phycobiliproteins and fluoresceinated microbeads will permit amplification of the fluorescent signal where cells have few PATr sites.
  • the long wavelength rhodamines which are S basically Rhodamine Green derivatives with substituents on the nitrogens, are among the most photostable fluorescent labeling reagents known. Their spectra are not affected by changes in pH between 4 and 10, an important advantage over the fluoresceins for many biological applications. This group includes the tetramethylrhodamines, X-rhodamines and Texas Red derivatives.
  • an amino group or groups in the polyamine are reacted with reagents that yield fluorescent products, for example, fluorescamine, dialdehydes such as ⁇ -phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.
  • reagents that yield fluorescent products for example, fluorescamine, dialdehydes such as ⁇ -phthaldialdehyde, naphthalene-2,3-dicarboxylate and anthracene-2,3-dicarboxylate.
  • 7-nitroben-2-oxa-l,3-diazole (NBD) derivatives are useful to modify amines to yield fluorescent products.
  • the most preferred reporter moiety is a fluorophore, either one which phosphoresces spontaneously or one which fluoresces in response to irradiation with light of a particular wavelength.
  • Chemiluminescent reporters are also acceptable. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • B. Radioactive Head Groups In order to use a probe for detection, various radioactive groups can be added for localization of the probe. Radioactive groups can be any of the various sulfonyl chlorides, acyl halides, or other activated groups or non-activated groups that contain a radioactive element which can be inco ⁇ orated into polyamine analogues. The polyamine itself can contain a radioactive element which may aid in detection.
  • Radioactive reporter moieties are detected by measurement of the emitted radiation.
  • suitable radioactive reporter moieties are those labeled with gamma -emitters such as 125 1, 1 3 I and 99m Tc. Some ⁇ and ⁇ emitters may suffice for detection.
  • the immobilization may be to any "solid support" capable of binding a capture protein (e.g., streptavidin or antibodies).
  • a capture protein e.g., streptavidin or antibodies.
  • Well-known supports, or carriers include glass, polystyrene, polypropylene, polyethylene, polyvinylidene difluoride, dextran, nylon, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the nature of the carrier can be either soluble to some extent or insoluble for the pu ⁇ oses of the present invention.
  • the support material may have any structural configuration so long as the immobilized molecule is capable of binding to its Hgand.
  • the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube or microwell, the external surface of a rod, or a chip.
  • the surface may be flat such as a sheet, test strip, microwell bottom, etc.
  • magnetizable groups such as gadolinium complexes or electron-opaque groups. Those skilled in the art will know many other suitable carriers for binding antibody or antigen, or will be able to ascertain such by use of routine experimentation.
  • the preferred embodiment of the PAT assay for identifying PAT inhibitors is a high throughput assay using a fluorescent polyamine-like probe. Transport of the probe through the cell membrane via a PATr is tested in the presence of candidate transport inhibitors. This and all subsequent steps are preferably performed by a robotic system for increased efficiency.
  • 96 well sterile microplates are plated in 96 well sterile microplates at an appropriate density for adherence and mid-log growth within 15-96 hours (the "test plate”).
  • a pre-plate (96 well plate format) is prepared using, for example, a combinatorial library of potentially inhibitory compounds (such as those prepared by PanLabs Inc., Bothell, WA). See also description of plyamine combinatorial libraries, below.
  • 1 nmole of the test inhibitor in each well 1 ⁇ mole of aminoguanidine and 1-100 nmoles of the fluorescent polyamine probe are added.
  • the final volume can be from about 25 ⁇ l to about 200 ⁇ l.
  • Aminoguanidine is included to prevent the oxi dative breakdown of the polyamine compounds in the culture medium.
  • the assay is initiated by transferring the contents of the pre-plate to the test plate in a timed fashion.
  • the cells are incubated with the test inhibitor and the fluorescent polyamine probe for 1 -60 minutes.
  • the incubation is terminated by removing the medium and washing the wells three times with ice cold medium containing 1 mM aminoguanidine and 1 ⁇ M spermidine.
  • the cells are then lysed with detergent ⁇ e.g. , 1 OO ⁇ l of 0.1 % sodium dodecylsulfate) and transferred to a fluorescence plate reading system, preferably a top reading fluorescence spectrophotometer.
  • a well is scored as positive if the test inhibitor causes the fluorescence to drop below 50% of the negative (no inhibitor) control.
  • the inhibitor compounds which score positive are then tested to determine their binding constants by repeating the above assay while varying the concentrations of both the fluorescent probe and the test inhibitor.
  • a standard kinetic analysis is performed to quantify the type of inhibition ⁇ e.g., competitive vs. noncompetitive) and its effectiveness.
  • the present inventors were able to screen about 20 compounds per week for the ability to inhibit PAT in a cell line. With the high throughput fluorescent assay disclosed herein, one can screen 250 compounds or more per week. Although the radiometric assay might lead to identification of the same compounds, the time scale would be much longer.
  • the preferred detection method is based on the fluorescence of the modified polyamine probe of the present invention.
  • other useful detection methods which include chemiluminescence, colorimetry as well as conventional radiometry.
  • chemiluminescence detection a chemiluminescent group is substituted for the fluorescent group.
  • fluorescent fluorescamine adducts can be converted to chemiluminescent products with bis-trichlorophenyl oxalate (Walters, DL et al,
  • colorimetric detection is used with chromophores having high extinction coefficients.
  • High throughput screening has become an essential part of the rapid drug discovery process.
  • HTS High throughput screening
  • many compounds can be assayed using candidate compounds from existing libraries or libraries synthesized by combinatorial approaches such as that described herein.
  • Compounds numbering in the thousands to hundred thousands are now routinely screened in the pharmaceutical industry, using microtiter plate formats with either 96 or 384 wells and robotic devices which transfer reagents, wash, shake and then measure activity signals which are directly imported in computer compatible form.
  • the robotic devices and optimization programs to aid rapid drug development are well-known art.
  • the three requirements of HTS are speed, accuracy and economy.
  • Radiometric Assays In a conventional radiometric assay, the cells are incubated under growth conditions with [5,8- ,4 C]spermine in serum-free medium for varying intervals. Cells are plated at a known density in 24 well plates in standard tissue culture medium and allowed to adhere and grow for 15-96 hours. Due to the low signal of the radiolabel in this assay, 96 or 386 well microplates cannot be used in this assay, creating a major bottleneck for throughput. Cell numbers are determined, and the plate is placed in a temperature controlled system at 37°C.
  • Aminoguanidine is added to the medium to a final concentration of 1 mM.
  • the inhibitor to be tested and the radioligand preferably 3 H-spermidine, 14 C- spermidine or ,4 C-spermine
  • the assay is initiated by the mixing of the inhibitor and radioligand.
  • the cells are incubated for an interval of about 1-60 minutes depending on cell type.
  • the assay is terminated by removing the medium and cooling the plates to 4°C.
  • the cells are then washed with cold medium three times, dissolved in 0.1% sodium dodecylsulfate, and the radioactivity in solution is determined by scintillation counting.
  • reporter moiety can be bound to a terminal amine or to an internal site of a polyamine. Most preferred are single substituted fluorescent groups bonded to any of the nitrogens. Substitution of fluorescent groups on the different carbons of a polyamine is exemplified by N-(4-dansylaminobutyl) spermine-5-carboxamide.
  • the number of reporter groups, e.g., fluorophores, per polyamine analogue may vary. The maximum number of fluorophores that allows competitive interaction with the PATr is preferred. In the case of monodansylspermine (MDS), the optimal number of fluorophores is one. If the reporter moiety is dansyl, the sensitivity of the assay can be improved by use of an anti-dansyl antibody (Molecular Probes, Eugene OR) during fluorescence measurement (in accordance with the manufacturer's instructions).
  • Preferred polyamine probes for the PAT assay have the following characteristics: they bind to the PATr, compete with the natural substrate ( Figures 27 and 28) and are internalized into the cell after such binding (Example XXV) .
  • a preferred probe comprises a fluorophore, a chromophore or a luminescer such as a chemiluminescer or a bioluminescer ⁇ Clin. Chem. 25:512, 1979) coupled to a polyamine core.
  • the amount of probe taken up in an assay, or the intracellular localization of the probe are determined by measuring and/or localizing the signal emitted by the reporter moiety after the probe has had the opportunity to bind to the PATr and be taken up.
  • the probe may be a specifically binding Hgand or one of a set of proximal interacting pairs.
  • Enzymatic Enhancement ELISA Another preferred approach to a sensitive screening assay uses the enzymatic amplification of the signal emitted by the detectable label on the polyamine probe.
  • a more specific example is a cofactor-labeled probe which can be detected by adding the enzyme for which the label is a cofactor and a substrate for the enzyme.
  • Such an enzyme is preferably one which acts on a substrate to generate a product with a measurable physical property such as color. Examples of such enzymes are listed below.
  • a preferred example of an enzymatically enhanced assay may be done by exploiting the biotin/streptavidin system (see Figure 31).
  • a biotinylated polyamine such as spermine is prepared, for example, as shown in Figures 29 and 30.
  • the PAT assay is performed as described above except that the probe in the appropriately lysed cells is detected using one of the formats described below.
  • the cells are lysed and the lysate filtered through negatively charged microplate membranes which retain the positively charged polyamine.
  • the membranes After washes and appropriate blocking of unreacted sites with a protein such as albumin, the membranes are incubated with a streptavidin-enzyme conjugate and washed.
  • the membranes are treated with a chromogenic substrate of the enzyme and incubated for a set time after which the color signal is read in a spectrometer. Colorimetric, fluorescent and chemiluminescent substrates are compatible with this procedure.
  • the lysate is reacted with a polylysine-treated plate using glutaraldehyde to crosslink the polyamine analogue to the plate.
  • the Schiff base is then reduced to produce an immobilized Hgand or analogue such as DACS.
  • the Hgand can then be detected through the appropriate reporter system.
  • the reporter is detected by anti-dansyl IgG antibody coupled to an anti-rabbit IgG conjugated to an HRP (horse radish peroxidase) detection system
  • An additional embodiment of the enzymatically enhanced assay is the use of an antibody to the reporter moiety such as the hapten 2,4-dinitrophenyl group
  • DNP 2,4,6-trinitrophenyl
  • the polyamine can be conjugated with different hapten groups (other than biotin).
  • biotinylated spermine derivatized with dansyl after transport into the cells and cell lysis, these molecules are immobilized to streptavidin-coated microplate wells and washed.
  • An antibody specific for dansyl is allowed to bind to any dansyl groups which have become immobilized.
  • the immobilized antibody is then allowed to react with a second antibody (an anti-immunoglobulin specific for the anti-dansyl antibody) conjugated to an enzyme.
  • a chromogenic substrate for the enzyme is added for a set interval.
  • the evaluation of the enzyme- generated signal is a measure of the amount of polyamine bound to the PATr.
  • enzymatic assays are in principle, similar to well-known enzyme immunoassay or enzyme-linked immunosorbent assay (ELISA). See, for example, Butler, J.E. (ed.) Immunochemistry of Solid Phase Immunoassay, CRC
  • Enzymes which can be conjugated to the streptavidin or antibody include, but are not limited to, horseradish peroxidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase,
  • staphylococcal nuclease ⁇ -V-steroid isomerase
  • yeast alcohol dehydrogenase ⁇ - glycerophosphate dehydrogenase
  • triose phosphate isomerase asparaginase, glucose oxidase, ⁇ -galactosidase, ribonuclease, urease, catalase, glucoamylase and acetylcholinesterase.
  • the biotin-spermine-hapten (preferably dansyl) molecules are captured using an immobilized anti-dansyl antibody.
  • two reporter molecules must be coupled to the polyamine analogue as in Figure 31.
  • One reporter is used for immobilization such as to an antibody- containing bead (or other solid support) to enable the washing away of excess probe.
  • the other reporter is used for sensitive detection. Streptavidin conjugated to an enzyme or streptavidin followed by enzyme-conjugated anti-streptavidin is preferred for signal amplification detection.
  • the linker between biotin and the polyamine is important. It should be designed to maximize binding with steptavidin after the polyamine-biotin has been immobilized to the solid support ⁇ Affinity Chromatography: Principles and Methods, Pharmacia, 1993).
  • a number of different linker molecules can be introduced between the polyamine and biotin, e.g., biocytin. Many different linkers of varying lengths are known in the art. The same considerations are important for ligands/labels other than biotin.
  • the present invention may also be used for histochemical or cytochemical localization of polyamines after uptake.
  • the polyamine analogues described herein localize within the cell. Probes of this type can therefore be used in cytochemical analyses of cells or tissues to identify cells or sites within cells with abnormally high or low levels of polyamines.
  • N'-dansylspermine was shown to localize specifically to the nucleoli and the nuclear membrane (Example XXVI).
  • the structure of the nucleus is a known indicator for the staging of progressing cancer.
  • the present fluorescent probes can be inco ⁇ orated into traditional
  • the reporter probe can be used to quantitate the binding of polyamines to known polyamine targets and binding sites. These sites include the NMDA receptor, K + inwardly rectifying channel
  • polyamines also bind specifically to RNA and DNA, and polyamine interactions play a role in hypusine synthesis. Due to the ease of developing modified polyamine analogues as described herein, specific analogues can be developed for each polyamine binding target.
  • proteins bind polyamines. Assays for such proteins are included in the scope of this invention and can identify drug candidates by competition assays using a bound fluorescent polyamine. In addition a tightly or irreversibly binding polyamine analogue can be used to extract and isolate any polyamine-binding protein or other polyamine-binding target of interest. Some proteins undergo conformational changes when a substrate or a polyamine is bound. The present screening assays are adapted so that the probe either binds or does not bind to a protein when the latter is in a given conformational state.
  • Protein Kinase A2 an isozyme that, upon bindin polyamine, undergoes a conformational change that modulates the enzyme's substrate binding site.
  • DACS 4 is one such compound, with a K ⁇ 10 nM.
  • DACS was tested as an inhibitor of cell growth ( Figure 22-24; Example XIX) in the presence of polyamines or an ODC inhibitor, DFMO.
  • R values were calculated as the ratio of the IC 50 in the absence of DFMO over the IC 50 in the presence of DFMO (Example XX).
  • the three compounds 6, 4 and 5 in Figure 2 (Example XX) had the best combination of K j 's (5, 10 and lO ⁇ M, respectively) and R values (220, 400 and 210, respectively) as summarized below:
  • Example XXI describes the inhibition of spermidine/spermine acetyl- transferase enzymatic activity by DACS ( Figure 26). Based on this, some of these compounds, if internalized, may serve a dual pu ⁇ ose.
  • Preferred compounds for use in pharmaceutical compositions include all of those mono- and di-substituted polyamine compounds described above, most preferably DACS (4) and compounds 5, 171 and 6, primarily in the form of pharmaceutically acceptable salts of the compounds.
  • Pharmaceutically acceptable acid addition salts of the compounds of the invention which contain basic groups are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids in the presence of the basic amine by methods known to the art.
  • Exemplary of the acid addition salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate,
  • the compounds of the invention possess the ability to inhibit PAT or polyamine synthesis, properties that are exploited in the treatment of any of a number of diseases or conditions, most notably cancer.
  • a composition of this invention may be active per se, or may act as a "pro-drug" that is converted in vivo to active form.
  • the compounds of the invention may be inco ⁇ orated into convenient dosage forms, such as capsules, impregnated wafers, tablets or injectable preparations. Solid or liquid pharmaceutically acceptable carriers may be employed.
  • the compounds of the invention are administered systemically, e.g., by injection.
  • injection may be by any known route, preferably intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.
  • injectables can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • Solid carriers include starch, lactose, calcium suifate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • sterile injectable liquid e.g., a solution
  • an ampoule e.g., an ampoule
  • aqueous or nonaqueous liquid suspension e.g., aqueous or nonaqueous liquid suspension.
  • the pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry involving such steps as mixing, granulating and compressing, when necessary for tablet forms, or mixing, filling and dissolving the ingredients, as appropriate, to give the desired products for oral or parenteral, including , topical, transdermal, intravaginal, intranasal, intrabronchial, intracranial, intraocular, intraaural and rectal administration.
  • the pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
  • the pharmaceutical composition may be administered topically or transdermally, e.g., as an ointment, cream or gel; orally; rectally; e.g., as a suppository, parenterally, by injection or continuously by infusion; intravaginally; intranasally; intrabronchially; intracranially intra-aurally; or intraocularly.
  • the compound may be inco ⁇ orated into topically applied vehicles such as a salve or ointment.
  • the carrier for the active ingredient may be either in sprayable or nonsprayable form.
  • Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water.
  • Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like. If desired, these may be sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • Preferred vehicles for non-sprayable topical preparations include ointment bases, e.g., polyethylene glycol-1000 (PEG-1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
  • peptide preferably in combination with a solid or liquid inert carrier material
  • aerosol preparations wherein the peptide, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and/or antioxidants in addition to the compounds of the invention.
  • an effective amount of the compound for the preferred topical applications, especially for humans, it is preferred to administer an effective amount of the compound to an affected area, e.g., skin surface, mucous membrane, eyes, etc.
  • an affected area e.g., skin surface, mucous membrane, eyes, etc.
  • This amount will generally range from about 0.001 mg to about 1 g per application, depending upon the area to be treated, the severity of the symptoms, and the nature of the topical vehicle employed.
  • compositions of the invention be given in combination with one or more additional compounds that are used to treat the disease or condition.
  • the polyamine derivatives are given in combination with anti- tumor agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate, pritrexim or trimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes or enzyme inhibitors, e.g., asparaginase; topoisomerase inhibitors, e.g., etoposide; or biological response modifiers, e.g., interferon.
  • pharmaceutical compositions comprising any known cancer therapeutic in combination with the substituted polyamines disclosed herein are within the scope of this invention. Most preferably, the present
  • compositions of the invention may also comprise one or more other medicaments such as anti-infectives including antibacterial, anti-funggal, anti-parasitic, anti-viral, and anti-coccidial agents.
  • anti-infectives including antibacterial, anti-funggal, anti-parasitic, anti-viral, and anti-coccidial agents.
  • Typical single dosages of the compounds of this invention are between about 1 ng and about lOg/kg body weight.
  • the dose is preferably is between about O.Olmg and about lg/kg body wt. and, most preferably, between about 0.1 mg and about lOOmg/kg body wt.
  • dosages in the range of about 0.01-20% concentration of the compound, preferably 1-5% are suggested.
  • a total daily dosage in the range of about 1 -500 mg is preferred for oral administration.
  • the foregoing ranges are, however, suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are expected.
  • Effective amounts or doses of the compound for treating a disease or condition can be determined using recognized in vitro systems or in vivo animal models for the particular disease or condition.
  • many art- recognized models are known and are representative of a broad spectrum of human tumors.
  • the compounds may be tested for inhibition of tumor cell growth in culture using standard assays with any of a multitude of tumor cell lines of human or nonhuman animal origin.
  • Many of these approaches, including animal models, are described in detail in Geran, R.I. et al, "Protocols for Screening Chemical Agents and Natural Products against Animal Tumors and Other Biological Systems (Third Edition)", Cane. Chemother. Reports, Part 3, 3:1-112, which is hereby inco ⁇ orated by reference in its entirety.
  • Such compounds are useful as drugs in a number of diseases, particularly against cancer. They are also useful as a component in combination drug therapy with, for example, a polyamine synthesis inhibitor such as DFMO (which inhibits ornithine decarboxylase, ODC) or with other agents, thereby providing novel combination chemotherapy regimens.
  • DFMO which inhibits ornithine decarboxylase, ODC
  • the compounds of the present invention are also useful for treating other diseases or conditions in which polyamines play a role as described above.
  • This invention extends the repertoire of chemistries available to explore SARs around drug targets for PAT inhibition and other relevant pharmacological and industrial targets.
  • a powerful combinatorial methodology is provided.
  • the direction of extension of the polyamine chain also ensures that over- alkylation does not occur while at the same time ensuring high yields.
  • an activated tert-alkoxycarbonyl MeO-PEG polymer is synthesized as indicated in Figure 34.
  • this polymer is reacted with a free amino- protected aldehyde extender synthon.
  • Figure 35 depicts the production of these extenders from either commercially available amino alcohols or the chiral amino acid precursor pool.
  • the synthetic cycle is followed using the reductive amination reaction with NaBH 3 CN initially to extend the backbone followed by an additional reductive amination step with an aldehyde to terminate the secondary amine produced above (Figure 36).
  • the final steps are the capping and acid cleavage from the polymeric support to provide the desired polyamine analogue as an acid salt (Figure 37).
  • MeO-PEG-OH Polyethylene Glycol Derivatives. Shearwater Polymers, Inc., 2307 Spring Branch Road, Huntsville, AL 35801
  • This material has the unique properties of (a) solubility in most commonly used organic solvents (see Table 1; Bayer, E. et al, In Proc. Eur. Pept. Symp, 13th, 129, 1975), and (b) precipitability by the addition of diethyl ether. These properties overcome many of the problems of solid-phase synthesis. For example,
  • the coupling method is ready to be tested and used.
  • the first extender unit 8a is reacted with the activated polymer to give the protected aldehyde linked through a carbamate, 9a ( Figure 36).
  • Selective deprotection of the 1,3-diphenylimidazolidine group from the terminal aldehyde of 9a produces the required starting substrate for the coupling reaction.
  • This hydrolysis, using a weak acid such as acetic acid poses no difficulties since the Boc-like linker moiety is expected to be cleaved under more strenuous acid conditions such as 10% TFA in CH 2 CH 2 .
  • aldehydic protecting groups including acyclic acetals, cyclic acetals or the acid stable dithio acetals may be inco ⁇ orated into the scheme.
  • the polymeric aldehyde 10a is then reacted with an excess of the next free amine/protected aldehyde subunit 8a (Figure 35) under the Borch reductive amination conditions in a suitable solvent such as THF or methanol (Borch, R.F. et al, J. Am. Chem. Soc. 93:2897-2904, 1971).
  • Reductive amination reactions on solid supports are well-known (Sasaki, Y. et al. J. Med. Chem.
  • a large series of simple straight chain or branched alkyl aldehydes is available. These modified polyamines would have greater lipophilic properties, thus increasing their ability to cross biological membranes and the blood-brain barrier. Any of a large number of unsaturated alkene aldehydes could also be used.
  • a particularly interesting example is acrolein, that can serve as a protecting group thereby allowing return to the secondary amine by deprotection of the resulting allyl amine with Rh(Ph 3 P) 3 Cl catalyst (Laguzza, B. C. et al, Tetrahed.
  • aromatic aldehydes A wide variety of aromatic aldehydes is also available. Any of a series of substituted benzaldehyde derivatives together with a variety of heterocyclic aromatic aldehydes could be used.
  • RNA polymers creates the potential for specific, tightly bound polycationic DNA "triple helix" analogues(Dempcy, R.D. et al, Proc. Natl. Acad. Sci. U.S.A, 93:4326-4330, 1996; Goodnow, Jr., R.A. et al, Tetrahedron Lett. 35:3195-3198, and 3199-3202, 1997).
  • Such a series would allow analysis of the molecular space surrounding the polyamine targets (described below).
  • the final capping and cleavage from the polymeric support are performed ( Figure 37).
  • the chemistry of the final capping is again very versatile.
  • the cleaved polyamine analogue 15a contains primary amino functions at both ends.
  • a reaction of the free terminal aldehyde with a primary amine is performed. Reaction with a secondary amine would yield a tertiary amine at the terminal position.
  • Another way of gaining a bifunctional terminal, tertiary amine is by reaction with a primary amine followed by reaction with an aldehydic terminator as above.
  • UV quantification Free amino groups are quantified by reaction with ninhydrin followed by UV analysis (Kaiser, E. et al, Anal. Biochem. 34:595, 1979). Infrared spectroscopy is used for functional group identification. Using the information from these techniques together with weights of the final products after cleavage from the polymer, correlations between the individual analytical methods are made. The final, cleaved products are thoroughly analyzed by standard techniques including: 'H- and 13 C-NMR, UV analysis where applicable, IR spectra, melting points, HPLC and TLC retention times and elemental analysis.
  • liquid-phase synthesis can also be performed using as solid-phase supports polystyrene resins, chip-based systems, multi-pin systems and microwells containing hydroxyl groups.
  • solid-phase supports polystyrene resins, chip-based systems, multi-pin systems and microwells containing hydroxyl groups.
  • Many solid supports with a hydroxyl linker are available, e.g., the Wang resin (Wang, S.-W.,
  • reaction conditions in the solid- phase approach are designed to give optimal yields and minimize side-reactions or incomplete reactions. Therefore, excess reagents are used and are washed away; unwanted reagents are also removable with scavenger resins (Booth R.J. et al, J. Am. Chem. Soc. 119: 4882, 1997). Catalytic resins can be used to speed up reactions.
  • FIG. 39 An example of a solid support with alternative linking group, to synthesize a polyamine analogue is shown in Figure 39, illustrating 3,4-dihydro-2H-pyran-2- yl-methoxymethyl polystyrene 16a (Calbiochem/Novabiochem, La Jolla, CA) (Thompson, L. A. et al, Tetrahed. Lett, 35: 9333, 1994). Reaction of 12 with the blocked aminoaldehyde yields 17a which is deblocked with HgCl 2 /HgO to aldehyde 18a.
  • Reaction of aldehyde 18a with a blocked aminoaldehyde in the presence of NaBH 3 CN yield 19a with an extended chain containing a secondary amine which blocked with a terminator aldehyde to yield 19a.
  • Compound 19a can either be deblocked and cleaved from the resin to yield a product or can be reacted in a next cycle.
  • the desired product containing a hydroxy-tail is released from the resin by treatment with 95% TFA/5% H 2 O.
  • the multipin-method is based on a modular 8x12 matrix of dimensionally stable polypropylene/polyethylene pins to which a graft polymer is covalently linked. Synthesis is performed upon the graft polymer, which can be varied to suit the application. Examples of multipin systems (Chiron Mimotopes, San Diego, CA) containing different linker groups are shown in Figure 40.
  • the Rink amide linker is shown as structure 23a coupled to the pin P (Rink H., Tetrahed. Lett., 25:1787- 1790, 1987). This linker requires the removal of the Fmoc protecting group prior to use. The desired product is then synthesized by building it out from the free amino group.
  • the product can be cleaved using 5% TFA/CH 2 Cl 2 to give primary amides.
  • This group is stable to weak acid and base.
  • Pins with structures 23a and 25a are used primarily for coupling carboxylic acids. The stability of 23a s good, and it is only labile to strong base and is cleaved with 95% TFA/H 2 O. In contrast 25a is stable to strong base and is cleaved with 95% TFA/H 2 O (Valerio, R.M. et al, Int. J. Peptide Protein Res. 44. 158-165, 1994).
  • Structure 24a is used to couple acids which can be cleaved with either NaOH or NH 2 R to give amides.
  • This pin type can be used in the presence of HF, TFA and weak base.
  • Structure 27a performs similarly to 25a, but is more suitable for milder acids and is generally more labile (Bray, A.M. et al, J. Org. Chem. 59:2197-2203, 1994).
  • Structure 26a is used to couple carboxylic acid, is cleaved following alkylation with CH 2 N 2 with NaOH or NH 2 R (to give amides) and is stable to strong acid and strong bases prior to alkylation. It will be evident to those skilled in the art that these solid supports can be inco ⁇ orated in the approaches described above. In addition, other solid supports known in the art can be combined with known chemistries to generate polyamine analogues containing different functional groups determined by the particular cleavable linker used.
  • N'-dansylspermine 3 Synthesis of N'-dansylspermine is illustrated in Figure 7. To 0.81 g (4 mmole) of spermine and 0.1 g (mmole) of triethylamine in 30 ml dry CH 2 C1 2 cooled down to 4C, was added dropwise 0.27 g (1 mmole) dansyl chloride dissolved in 20 ml dry CH 2 C1 2 over 90 minutes. The temperature was allowed to
  • the material was dissolved in 8 ml 1.0 M ammonium acetate pH 7.4 and was chromatographed on a Biorad 70 weak cation exchanger (1.5 x 48 cm) using a pH gradient between 1.0 M ammonium acetate and 1.25 M hydrochloric acid over
  • N'-(l-pyrenylsulfonyl)spermine 15 Synthesis of N'-(l-pyrenylsulfonyl)spermine 15 is illustrated in figure 5.
  • the material was dissolved in 8 ml 1.0 M ammonium acetate pH 7.4/MeOH 1 : 1 and was chromatographed on a Biorad 70 weak cation exchanger (1.5 x 48 cm) using a pH gradient between 1.0 M ammonium acetate pH 7.4 and 1.25 M hydrochloric acid/methanol (1 :1) over 500 ml with a flow rate of 0.5 ml per minute, collecting 8 ml fractions. Fractions containing a single spot were collected, adjusted to pH 10.5 and extracted with 2 x 25 ml ethyl acetate. This ethyl acetate fraction was filtered through Whatman filter paper and evaporated to dryness.
  • N'-((l-carbonyl)-4-(l-pyrenyl)butane)spermine 37 Synthesis of N 1 -(( 1 -carbonyl)-4-( 1 -pyrenyl)butane)spermine is illustrated in Figure 6. To 0.29 g (1 mmole) of 1 -pyrenebutyric acid dissolved in CHC1 3 with heating were added 0.19g (1 mmole) of EDC 0.12g (1 mmole) of N- hydroxysuccinamide and was stirred at room temperature for 30 minutes when this solution was added drop-wise to 0.82 g (4 mmole) spermine dissolved in 20 ml CHC1 3 .
  • N-( 1 -anthracenyl)-N ' -(N 1 -spermidyl)urea (9) Synthesis of N-(l-anthracenyl)-N'-(Nl-spermidyl)urea is illustrated in Figure 4.
  • N-(l-anthracenyl)-O-(p-nitrophenyl)urea (1.6g ; 86% yield) was filtered from the cooled reaction and washed with benzene. This product was used without further purification.
  • n 1 to 10.
  • n 1 to 10.
  • Disubstituted functionalizable compounds are well known in the art, for example sulfonyl chlorides, benzoyl chlorides, cyanates, thiocyanates, etc.
  • sulfonyl chlorides for example sulfonyl chlorides, benzoyl chlorides, cyanates, thiocyanates, etc.
  • cyanates for example sulfonyl chlorides
  • thiocyanates thiocyanates
  • N'-[6-aminocaproylspermine] 171 This reaction scheme is carried out as described in detail below.
  • reaction vials In each of three 10 ml reaction vials (React-VialTM Pierce, Rockford, IL) were placed 0.74 mmol of spermine and 0.15mmol of triethylamine. Similarly in three additional reaction vials were placed 0.74 mmol of spermine and 0.15mmol of triethylamine. Similarly in three additional reaction vials were also placed 0.74 mmol of putrescine and 0.15 mmol triethylamine.
  • Each vial contained also an exhaust consisting of a 2.5 ml syringe filled with anhydrous CaCl 2 with out the plunger.
  • the reactions were allowed to proceed for 16 hours at ambient temperature when it was extracted 2x2.5ml 5% sodium carbonate solution followed by 2x 2.5 ml
  • the present investors have developed a growth assay to use in screening for transport inhibitors that are synergistic with ODC inhibitors.
  • the estrogen insensitive human breast carcinoma MDA-MB-231 cell line as the primary cell line in the screen. This cell line, as with many breast cancers, has a high rate of polyamine transport ⁇ Anticancer Res. ⁇ 1991 ) 11 : 1807- 1814).
  • 1.0 ⁇ M spermidine was added to media to reverse the effects of ODC inhibitors.
  • the assay was also performed over seven days because this allows for the greatest dynamic range in cell growth due to the mechanism of ODC inhibitors. Cells need to divide several times before the intracellular level of polyamines begin to decrease to growth inhibitory levels. Therefore, growth does not significantly cease until the third to fourth day.
  • the growth assay alone does not verify a reduction of polyamine uptake. Therefore, the growth assay and a kinetic transport assay have been used to validate transport inhibition.
  • DACS is Effective in the Presence of Natural Polyamines Extracellular spermidine, spermine and putrescine can reverse the effects of ODC inhibitors through increased uptake into the cell.
  • the major excreted forms of polyamines (N'-acetylspermine and N'-acetylspermidine) can also reverse the effect of ODC inhibitors.
  • DACS prevents the natural polyamines, putrescine, spermidine, N'-acetylspermine and N'-acetylspermidine, from rescuing the cells from ODC inhibition. This is significant for several reasons. Reports in the literature suggest that there are more than one transporter. If this is true, DACS is effective at blocking the uptake of all of the polyamines at low concentrations.
  • DACS was tested in vitro in combination with ODC inhibitors against several human cancer cell lines. These include T-cell acute lymphoblastic leukemias (ALL), glioblastomas, prostate, and colon cell lines. DACS is effective against all these tumor cell lines in vitro.
  • Figure 24 shows the effects of DACS on ALL
  • PC-3 prostate cancer cells PC-3 prostate cancer cells.
  • R is the IC 50 for polyamine alone relative to the IC 50 for the polyamine analogue combined with an ODC inhibitor. This value of R, indicates the relative level of "synergism" between the polyamine analogue and ODC inhibitor. Under the growth assay conditions, the ODC inhibitor alone shows no inhibition.
  • DACS inhibited spermidine/spermine acetyltransferase (SSAT) in a dose-related manner.
  • Poly lysine plates were prepared by addition of 200 ⁇ l of polylysine (5 ⁇ g/ml) in 10 mM Tris-HCl buffer, pH 8.5, containing 10 mM NaCl and 10 mM
  • %Q NaN 3 The plates were incubated for 20 min at 37°C when the wells were washed 3X with 200 ⁇ l water. The plates were then treated with ⁇ l of 2.5% glutaraldehyde in 50 mM borate buffer pH 10.0 for 1 hr at 25°C, when the wells were washed with 200 ⁇ l of 50 mM borate buffer pH 10.0 twice and once with water. Various concentrations of either Nl-dansylspermine or DACS were added to the wells ranging between 0.1 and 10 pmoles/ well and incubated for 1 hr at room temperature. The plates were then washed with twice with 200 ⁇ l or PBS.
  • Enzyme activity was determined using either 100 ⁇ l of NBT or OPD (5 mg OPD/10 ml of 0.1 M citrate buffer, pH 5.0) and an incubation period of 10 minutes at room temperature. The color was measured at 630 nm in a plate reader.
  • This method is an alternate embodiment of the of the PAT assay using indirect detection to enhance the signal and lower the detection limits. This method allows for the detection of extremely low concentrations of probe.
  • This technology provides an approach for solving the problem of triple- helix forming antisense oligonucleotides (Chan, P.P. et al, J. Mol Med. 75: 267- 282 (1997) by combining the transportability of polyamines into cells with structural features of nucleotide sequence specificity.
  • the transport overcomes the limitations of bioavailability while also enhancing the bio-stability of such an oligomer.
  • Rl and R3 are both hydrogen.
  • the keto-function can also be a part of a ring structure.
  • R 2 and R 4 can be aliphatic, alicyclic, aromatic and heterocyclic.
  • Examples of compounds that could be contain aldehyde, ketone, amino-aldehyde or amino-ketone functions are dibenzofuran, acridine, 2,1,3-benzothiodiazole, quinoline, isoquinoline, benzofuran, indole, carbazole, fluorene, 1,3-benzodiazine, phenazine, phenoxazine, phenothiazine, adamantane, camphor, piperidine, alkylpiperazine, mo ⁇ holine, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, thiophene, furan, pyrrole, alkyl- 1,2-diazole, alkylimidazole, alkyl- 1H- 1,2,3-triazol, alkyl- 1 HI, 2,3, 4-tetrazole, thiazole,
  • a library of compounds is synthesized by using the appropriate blocked aminoaldehydes, aldehydes or ketones selected from commercially available sources or from synthetic routes known in the art.
  • Aminoaldehydes are synthesized in a variety of ways from various starting materials such as L-and D- amino acids, aminoalcohols, or alcohols or carboxylic acid substituted with NO 2 or -CN groups.
  • Aminoaldehydes are synthesized from appropriately blocked aminoalcohols by known procedures (Larack, R., In: Comprehensive Organic Transformations, VCH Publishers, Inc., NY, 1989, pp. 604-616).
  • Aminoaldehydes are directly synthesized from appropriately blocked aminocarboxylic acids or blocked aminonitrile ⁇ supra at p. 616-617).

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Abstract

La présente invention concerne des inhibiteurs du transport de la polyamine présentant des constantes d'inhibition inférieures de deux ordres de grandeur à celles des composés connus. Ces analogues de la polyamine constituent des agents pharmaceutiques utiles pour le traitement des maladies où l'on désire inhiber le transport de la polyamine ou d'autres protéines liant la polyamine, par exemple le cancer ou les lésions consécutives à une angioplastie. L'invention se rapporte à de nouveaux procédés chimiques synthétiques permettant d'obtenir des analogues de la polyamine, y compris à la production d'une bibliothèque combinatoire de polyamines. Ces procédés permettent d'obtenir des analogues présentant des activités désirables pour le diagnostic, les analyses de recherche et la thérapie. Les analyses de la présente invention sont utilisées pour le criblage à grand débit de cibles permettant de découvrir des médicaments qui interagissent avec le système de la polyamine.
PCT/US1998/014896 1997-07-15 1998-07-15 Nouveaux analogues de polyamine utilises comme agents therapeutiques et diagnostiques WO1999003823A2 (fr)

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AU84968/98A AU758570B2 (en) 1997-07-15 1998-07-15 Novel polyamine analogues as therapeutic and diagnostic agents
US09/341,400 US6172261B1 (en) 1997-07-15 1998-07-15 Polyamine analogues as therapeutic and diagnostic agents
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US09/396,523 US7208528B1 (en) 1997-07-15 1999-09-15 Polyamine analogues as therapeutic and diagnostic agents
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WO1999021542A2 (fr) * 1997-10-27 1999-05-06 The Regents Of The University Of California Procedes pour moduler la proliferation des macrophages au moyen d'analogues de polyamine
WO2000035941A2 (fr) * 1998-12-16 2000-06-22 Millennium Pharmaceuticals Limited Procede de preparation de polyamines
WO2000046187A2 (fr) * 1999-02-05 2000-08-10 Oridigm Corporation Modulateurs d'antizyme et leur utilisation
EP1085011A1 (fr) * 1999-09-15 2001-03-21 Oridigm Corporation Polyamine analogues comme agents therapeutiques et diagnostiques
WO2001072685A2 (fr) * 2000-03-24 2001-10-04 Oridigm Corporation Polyamine analogues, agents cytotoxiques
WO2001092218A2 (fr) * 2000-05-31 2001-12-06 Oridigm Corporation Nouveaux analogues de polyamine a utiliser en tant qu'agents therapeutiques et diagnostiques
EP1212619A1 (fr) * 1999-09-14 2002-06-12 Xenoport, Inc. Substrats et methodes de criblage pour proteines de transport
JP2002543163A (ja) * 1999-04-30 2002-12-17 スリル バイオメディカル コーポレイション 癌および前立腺疾患のための治療としての結合体
WO2003018014A2 (fr) * 2001-08-23 2003-03-06 The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Procedes d'inhibition de la formation de canaux vasculaires et procedes d'inhibition de la proliferation
WO2003066037A1 (fr) * 2002-02-05 2003-08-14 University Of Southampton Derives de spermidine destines au traitement de maladies neurodegeneratives chroniques
EP1373185A2 (fr) * 2001-01-08 2004-01-02 Mediquest Therapeutics, Inc. Analogues de polyamine hydrophobe et procedes d'utilisation correspondants
WO2005120451A1 (fr) * 2004-06-14 2005-12-22 Unilever Plc Procede de diminution de la fabrication de sebum et du diametre des pores
US7160923B1 (en) 1997-07-15 2007-01-09 Mediquest Therapeutics, Inc. Polyamine analogues as therapeutic and diagnostic agents
US7208528B1 (en) 1997-07-15 2007-04-24 Mediquest Therapeutics, Inc. Polyamine analogues as therapeutic and diagnostic agents
WO2007143735A2 (fr) * 2006-06-07 2007-12-13 The University Of Kansas Sulfonamides polycationiques et utilisation de ceux-ci
US7432302B2 (en) 2001-01-08 2008-10-07 Mediquest Therapeutics, Inc. Composition containing polyamine transport inhibitor and use thereof
EP2096106A1 (fr) * 2006-11-15 2009-09-02 Nihon University Nouveaux dérivés de polyamine
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JP4044728B2 (ja) 2008-02-06
US7160923B1 (en) 2007-01-09
US6172261B1 (en) 2001-01-09
WO1999003823A3 (fr) 1999-04-08
AU758570B2 (en) 2003-03-27

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